Two procyanidin fractions, namely oligomers and polymers isolated from grape seed methanolic extract were characterized. Phenolic composition and procyanidin purity of these fractions were determined by normal-phase and reverse-phase HPLC, thioacidolysis-HPLC, ESI-MS analyses, formaldehyde-HCl precipitation and elemental analysis. Antioxidant activities of these fractions and other well-known antioxidants were measured using xanthine-xanthine oxidase system for generating superoxide radical ({O2(-)}), the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method and the Fenton system for generating hydroxyl radical (HO). The results showed that both oligomeric and polymeric procyanidin fractions were highly pure, with the degree of polymerization ranging from 2 to 17-18 and 12 to 32-37, respectively. On the basis of molar concentration, polymeric procyanidins appeared the highest antioxidant activities, followed by oligomeric procyanidins, whereas catechins presented a lower antioxidant activity than its oligomers and polymers. These results indicate that the antioxidant activities of grape seed procyanidins are positively related to their degree of polymerization. Moreover, grape seed procyanidins presented higher antioxidant activities than other well-known antioxidants such as vitamin C, suggesting that grape seed procyanidins might be of interest to be used as alternative antioxidants.

In Hereditary Diffuse Gastric Cancer syndrome, E-cadherin germline mutations of the missense type harbour significant functional consequences. In this study, we have characterised the effect of T340A, A617T, A634V and V832M E-cadherin germline missense mutations on cell morphology, motility and proliferation. Wild-type E-cadherin and A617T expressing cells have an epithelial-like morphology, with polarised cells migrating unidirectionally. T340A and A634V expressing cells, fibroblast-like, have a high motile phenotype. We show that this phenotype is dependent on an increased level of active RhoA. V832M expressing cells grow in piled-up structure of round cells, as an effect of the disturbance of the binding between alpha-catenin and beta-catenin. The destabilisation of the adhesion complex is shown to hamper the motile capabilities of these cells. We did not observe any effect of the E-cadherin mutations on cell proliferation. We show the existence of a genotype-phenotype correlation between different E-cadherin mutations and cell behaviour. However, we demonstrate that the ability of cells expressing the different E-cadherin mutations to invade is independent on their motile capabilities, providing evidence that motility is neither necessary nor sufficient for cells to invade. Our data give new insights into the understanding of the mechanisms linking invasion and E-cadherin mutations in diffuse gastric cancer.

No abstract

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF-LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF-LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a b-catenin-dependent activation of the WNT pathway. Oncogene (2003Oncogene ( ) 22, 5716-5719. doi:10.1038 Keywords: E-cadherin; hereditary diffuse gastric cancer; mutations; cell-cell adhesion; invasion; TCF-LEF E-cadherin is a transmembrane glycoprotein localized at the adherens junctions of epithelial cells, where it mediates homophilic, Ca 2 þ -dependent cell-adhesion (Shore and Nelson, 1991;Shapiro et al., 1995;Pertz et al., 1999). The adhesion process is accomplished by homophilic interactions between extracellular E-cadherin domains, leading to the formation of zipper-like structures (Pertz et al., 1999). The E-cadherin intracellular domain interacts with catenins, assembling the celladhesion complex. b-Catenin and g-catenin compete for the same binding site at the E-cadherin cytoplasmic tail, directly linking the adhesion complex to the cytoskeleton through a-catenin (Christofori and Semb, 1999). These interactions represent a prerequisite not only for cell-cell adhesion, but also for inhibition of cell motility and invasion (Chen et al., 1997). The dual role of b-catenin, as a component of the cell-cell adhesion complex as well as transcriptional coactivator in the WNT growth factor signal pathway has been extensively characterized (Cavallaro et al., 2002). In the absence of WNT, phosphorylation by glycogen synthase kinase-3b (GSK-3b) targets b-catenin for degradation in the adenomatous polyposis coli (APC)-GSK-3b complex. It was reported that APC and/or GSK-3b inactivation as well as mutations of AXIN and bcatenin itself (Bienz and Clevers, 2000) leads to an accumulation of high level of b-catenin into the cytoplasm; b-catenin could translocate into the nucleus and bind to a member of the TCF-LEF-1 family of transcription factors, activating gene expression (Polakis, 2000).Aberrant E-cadherin protein expression has been reported in several types of human cancer (Becker et al., 1994;Gayther et al., 1998;Guilford et al., 1998;Machado et al., 2001;Van Aken et al., 2001;Thiery, 2002). Despite what has been observe...